Identification of autonomous replication sequences in genomic and mitochondrial DNA of Crithidia fasciculata

Molecular and Biochemical Parasitology, 10 (1984) 151-160 Elsevier

151

MBP 00389

I D E N T I F I C A T I O N O F A U T O N O M O U S R E P L I C A T I O N S E Q U E N C E S IN G E N O M I C AND M I T O C H O N D R I A L DNA O F C R I T I I I D I A F A S C I C U L A T A

RAJU KODURI and DAN S. RAY* Department of Biology and Molecular Biology Institute, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024, U.S.A.

(Received 15 February 1983; accepted 30 June 1983)

High frequency transformation of Saccharomyces cerevisiae was used as a functional assay to isolate autonomous replication sequences(ars) from the genomicand kinetoplast DNA of the insect trypanosomatid Crithidiafascieulata. Three independent cloned genomic sequencesand one kinetoplast DNA sequence promoted high frequencytransformation and extrachromosomal maintenance of the YIp5 plasmid DNA in yeast. The kinetoplast DNA clone was sub-cloned to further localize the DNA sequence essential for ars activity. This element was shown to be contained in a 2 kb HindlII-EcoRI fragment derived from a 8 kb HindlII fragment of the maxicirclecomponent of the kinetoplast DNA. This 2 kb fragment is within a DNA sequence that has been shown to strongly hybridize to Trypanosoma brucei maxicircle DNA. Key words: Origin of replication; Autonomous replication sequence; Kinetoplast DNA

INTRODUCTION K i n e t o p l a s t i d flagellates possess several m o r p h o l o g i c a l a n d biochemical features, a m o n g which is the cell's single m i t o c h o n d r i o n , called the kinetoplast [1]. The kinetoplast c o n t a i n s a large n e t w o r k of D N A ( k D N A ) a b o u t 95% of which is comprised of small covalently closed circles called minicircles [see refs. 2 a n d 3 for recent reviews]. The r e m a i n i n g 5% of the D N A n e t w o r k is in the form of much larger maxicircles. The maxicircles are the f u n c t i o n a l equivalent of m i t o c h o n d r i a l D N A in other species. Very little is k n o w n a b o u t the o r g a n i z a t i o n of c h r o m o s o m e s in the kinetoplastid nucleus, the c h r o m o s o m e n u m b e r or the proteins that control D N A replication. A first step towards u n d e r s t a n d i n g the m e c h a n i s m s that regulate D N A synthesis in these p r o t o z o a w o u l d be to study the complexity, structure a n d organization of D N A replication origins in the c h r o m o s o m e a n d kinetoplast D N A s a n d their relationship to the events in the cell cycle. * To whom correspondence should be addressed. Abbrevianons: ars, autonomous replication sequence; kb, ktlobase pairs; kDNA, kinetoplast DNA; ccc, covalently closed circular; oc, open circular.

0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.

152 Integrative plasmids containing a selectable gene transform the yeast Saccharomyces cerevisiae at low frequency. By cloning random fragments of yeast DNA into such plasmids Struhl et al. [4] and Hsiao and Carbon [5] have identified DNA sequences capable of autonomous replication. Plasmids carrying autonomously replicating sequences (ars) transform yeast at a high frequency (up to 10 000 colonies per lag). It has been speculated further that the ars elements are normally used for initiation of yeast DNA replication based on the following observations: (i)'a majority of replicating yeast 2 lain plasmid DNA molecules were found to have initiated within a single ars sequence [6] and (ii) only plasmid DNA molecules containing a functional ars element initiated replication at a specific site in an in vitro replication system [7]. It has since been found that specific DNA fragments from a wide variety of other eukaryotes promote high frequency transformation of yeast [8]. These results suggest the possibility that the initiation of DNA replication occurs at specific sequences and that these sequences migh be similar in other eukaryotes. This notion is supported by the recent finding that a DNA sequence from the slime mold Dictyostelium discoidium that acts as an ars in yeast is needed to transform D. discoidium with plasmid DNA at high frequency [9] although it has not yet been shown that this ars mediates autonomous replication in D. discoidium. We have successfully used the differential transformation by hybrid plasmids with and without ars function in yeast to clone and isolate autonomously replicating sequences from the insect kinetoplastid Crithidia fasciculata. Both chromosomal DNA and the maxicircle component of k D N A were found to possess ars elements. MATERIALS AND METHODS Strains. Escherichia coli RL107 (leu, met, r k, m~) and E. coli RL108 (Tet a, recA56, leu, met, r~, m[) were used as recipients for the amplification of hybrid plasmids. S. cerevisiae NNY-1 (met, trp 1, ura3-52, his3-1, gal2, gall O, cir°) was used as a recipient in yeast transformation. Plasmids. The plasmid YIp5 is a derivative of pBR322 which carries the S. cerevisiae ura3 gene [4] but lacks the capacity for autonomous replication in yeast. YRpl2 is an ars÷ derivative of YIp5 and contains the yeast arsl sequence [8]. DNA isolation. Kinetoplast and chromosomal DNA from C.fasciculata was isolated according to the procedures described by Simpson et al. [10]. The procedure for extracting low molecular weight yeast DNA was that of Zakian et al. [11]. Plasmid DNA was isolated from E. coli cleared lysates [12] by centrifugation in CsCI/EtBr gradients. Transformation procedures. E. coli cells were transformed by the CaCI2 method [13]. Yeast cells were made competent for transformation by treatment with lithium acetate [14].

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Restriction endonucleases. Restriction enzyme digestions were usually carried out under conditions recommended by the distributor. The enzymes EcoRI, HindlII and T4 ligase were obtained from Bethesda Research Laboratories. Gel electrophoresis. Digested DNA samples were electrophoresed in 0.7% agarose horizontal slab gels in a Tris-borate-EDTA buffer as previously described [15]. Gel blotting and hybridization. DNA fragments were transferred from agarose gels to nitrocellulose filter paper by the method of Southern [ 16] and hybridized with pBR322 DNA labeled with 32p by nick translation as described by Rigby et al. [17]. After washing and drying, the filter paper was subjected to autoradiography on Kodak XR-1 film. RESULTS

Screening of C. fasciculata chromosomal and kinetoplast DNA for autonomously replicating segments. 10 ~tg of C. fasciculata chromosomal DNA was digested with EcoRI and ligated to 2 I.tg of YIp5 vector DNA cut with EcoRI. The hybrid plasmid pool was then used to transform E. coli RL108 in order to amplify the chimeric plasmid DNA. Approximately 2 )< 104 individual ampicillin-resistant transformants were pooled, grown in mass culture and the DNA purified by CsCI-EtBr density gradient sedimentation. The purified plasmid DNA was used to transform yeast strain NNY- 1 selecting for complementation of the strain's ura3 mutation. In this experiment 10 lag of chimeric plasmid DNA yielded 410 Ura ÷ colonies while the vector DNA alone yielded only 6 Ura ÷ colonies. Individual Ura ÷ isolates were picked and further examined to confirm the presence of ars elements. We adopted a different strategy for cloning maxicircle fragments from kinetoplast DNA. Since the HindlII digestion of k D N A releases only eight maxicircle fragments [18] plus minicircles, we chose to isolate hybrid plasmids representing each of the eight kDNA fragments and individually screen them for their ability to replicate autonomously in yeast. 20 lag of kDNA and 5 lag of YIp5 vector were cut with HindlII and ligated with T4 DNA ligase. 5 lag of the ligated pool was used to transform E. coli RLI07. The hybrid plasmids that were Amp R and Tet s were selected and screened for insert size using a rapid minilysate procedure [12]. Examination of hybrid plasmids from 56 independent clones revealed that all the HindlII generated kDNA maxicircle fragments were represented in the plasmid pool except for one 15 kb A-T rich HindlII fragment. Attempts to reclone the 15 kb HindlII fragment and a much larger 23 kb SalI fragment overlapping the 15 kb HindlII fragment resulted in an array of plasmids with extensive internal deletions. Upon transformation into yeast, none of the plasmids carrying HindlII fragments of maxicircle DNA nor any of ten selected clones containing HindlII-cleaved minicircles gave high frequency transformation except for the plasmid pCFK8. This plasmid

154 was f o u n d to c o n t a i n t w o HindlII i n s e r t s o f 8.0 k b a n d 2.5 k b a n d c o n f e r r e d the a b i l i t y o n Y I p 5 to t r a n s f o r m yeast at a high f r e q u e n c y (2.4 × 102 t r a n s f o r m a n t s per Ixg).

Evidence for extrachromosomal maintenance of chimeric plasmid DNAs in yeast. T h e e x t r a c h r o m o s o m a l existence o f these c h i m e r i c D N A s has b e e n e s t a b l i s h e d b y electrop h o r e t i c a n a l y s i s o f the D N A c o n t a i n e d in lysates o f ars÷yeast t r a n s f o r m a n t s . F i r s t , p l a s m i d D N A was e x t r a c t e d f r o m yeast cells t r a n s f o r m e d b y p l a s m i d s c o n t a i n i n g

EH

Pv~--------------~ B let kDN._____~A

a m p ( ( t YIpS, ~)~S

+x...I

v

um3

nuclea.~__~r DNA

x , , ~ Ec°RI

I

~[T4licjase

T4 ligase

E

tel

ura3 ~ EcoRI

uro3

~T4 ligose amp~ uro3 Fig. 1. Construction of Ars÷ chimeric plasmids. Purified kDNA or nuclear DNA of C. fasciculata was cleaved either with HindlIl or EcoRI, respectively, and cloned into the corresponding single restriction sites of the yeast plasmid YIp5. The plasmid YIp5 is derived from the E. coliplasmid pBR 322 and contains the S. cerevisiaeura3 gene. The Ars÷kDNA clone pCFK8 was reduced in size by deletion of two EcoRIfragments to yield plasmid pCFKI6. The Ars÷ nuclear DNA clones pCFNI, 2 and 3 contain single EcoRl fragments. The yeast ura3 gene is shown in black and C.fasciculata DNA is indicated by stippling. Restriction enzyme sites for the enzymes EcoRl (E), HindllI (H), BamH1 (H), Sail (S) and Pstl (P) are shown. Subscripts indicate specific sites on the maxicircle physical map of Hoeijmakers et al. [18].

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a

YRp12 OC

YRp12

b

c

d

-

C C C-

Fig. 2. Detection of plasmid DNAs in yeast lysates by hybridization with a ~2P-labeledpBR322 DNA probe. DNA was isolated from yeast clones transformed either by pCFK8 or pCFN 1, electrophoresed on a 0.7% agarose gel, transferred to nitrocellulose paper and hybridized to 32P-labeled pBR322 DNA as described [ 16]. A lysate of yeast carrying the replicative yeast plasmid YRp 12 (6.9 kb) was run in parallel as a control. Lane (a), YRpl2; Lane (b), pCFKS; Lane (c), an independent isolate of pCFKS; Lane (d) pCFNI.

156 either chromosomal or kDNA ars sequences. Each DNA was run on agarose gels, transferred to nitrocellulose filters and hybridized to nick-translated pBR322 DNA. All of the ars ÷ yeast transformants screened yielded discrete bands of plasmid DNA migrating slower than the YIp5 vector DNA. Fig. 2 shows the results for the plasmids pCFK8 and pCFN 1. Two distinct hybridizable bands representing covalently closed DNA (CCC-DNA) and open circular DNA (OC-DNA) are observed in each case. These species comigrate with the corresponding components from plasmid DNAs propagated in E. coli (data not shown). Size analysis o f ars ÷plasmids. Plasmid DNA from ten ars ÷genomic clones and two ars ÷ kDNA clones were used to transform E. coli to ampicillin resistance. Each DNA was

isolated by a rapid minilysate procedure and screened with regard to size on agarose gels. The plasmids from the ten genomic clones appeared to fall into three size classes. This apparent non-random size distribution possibly reflects a selection that occurred during amplification of the DNA library in E. coli prior to the yeast transformation and subsequent isolation of siblings. A representative plasmid of each size was selected for further study. These plasmids have been termed pCFNI (8.3 kb), pCFN2 (9.5 kb) and pCFN3 (12.6 kb). The insert sizes for each of the selected clones was determined by cleavage of each plasmid DNA with either HindIII (maxicircle DNA clone) or EcoRI (nuclear DNA clones) and electrophoresis on agarose gels. The HindIII digest of pCFK8 yields two fragments of 2.5 kb and 8.0 kb in addition to the 5.5 kb linear form of the vector YIp5 (Fig. 3, lane c). Similar results were obtained from HindIII digests ofE. coli RL108 minilysates from two independent isolates of pCFK8 that had been propagated in yeast and then reintroduced back into E. coli RL108 (Fig. 3, lanes a and b). This result suggests that no substantial deletions or rearrangements of the cloned kDNA segment is required for expression of the ars function. Each of the genomic clones contained only a single inserted EcoRI fragment (Fig. 3, lanes d-g). The inserts in pCFNI, pCFN2 and pCFN3 were determined to be 2.8, 4.0 and 7.1 kb in size. Subcloning o f the maxicircle ars element. The 8.0 kb insert (H3-H4) in pCFK8 has been

identified with fragment D3-D4 on the maxicircle physical map [18] based on the analysis of restriction digests using the enzymes Sail, SstI, XhoI and EcoRI (data not shown). To further localize the ars element, pCFK8 DNA was digested with EcoRI, religated and used to transform yeast selecting again for Ars÷transformants. Among the clones obtained, the smallest one (pCFK16) had deleted fragments E3-E4 and E4-E (Fig. 1). Fig. 4 shows the gel analysis of restriction digests of plasmids pCFK8 and pCFK 16. These results indicate that the entire 2.5 kb HindlII fragment and 6.0 kb of the 8.0 kb H i n d l I I fragment can be deleted without loss of ars activity. The remaining H i n d l I I - E c o R I fragment H3-E3 corresponds to fragment D3-R~ on the maxicircle physical map of Hoeijmakers et al. [18].

157

abc

d

•

f

g

h

kb

8.0

--

5.5-

2.5;

Fig. 3. Restriction enzymedigests of chimeric plasmid DNAs. Samples were digested and electrophoresed on 0.7% agarose gels as described in Materials and Methods. HindllI digests of minilysates of two independent clonescarryingpCFK8 whichhad been propagatedin yeastand then reintroducedback into E. coli RL108 (a) and (b) and purified pCFK8 DNA (c). EcoRI digestsof(d) YIp5,(e) pCFN 1, (f) pCFN2,and (g) pCFN3 and a Hindlll digest of Ad5 viral DNA (h) to provide molecular weight markers.

DISCUSSION We have used high frequency transformation of yeast as a functional selection for possible replication origins from genomic and kinetoplast D N A of the lower trypanosome C. fasciculata. The chimeric plasmids containing Crithidia D N A conferred self-replicating ability on the non-replicating yeast plasmid YIp5 and rescued the S. cerevisiae ura3 mutation. Of ten genomic clones rescued in yeast, three size classes of 8.3 kb, 9.5 kb and 12.6 k b were observed. The observation of distinct size classes of these genomic clones suggests that specific ars elements may have been enriched during amplification of the D N A library in E. coli. The kinetoplast D N A from C. fasciculata is composed of 5 000-10 000 2.5 kb minicircles and 10-30 36 kb maxicircles. Even though minicircles represented the largest single class of k D N A clones, consistent with their great abundance in the k D N A network, we failed to obtain Ars ÷ minicircle clones, Several cloned minicircles were tested for their ability to replicate autonomously in yeast but none were found to have significant ars activity. In contrast, the maxicircle plasmid pCFK8 expressed a very strong ars activity in yeast. The 8 kb maxicircle fragment corresponds to the H i n d l I I fragment D3-D4 on the maxicircle physical map of C. fasciculata and C.

158

a b

cd

e

f

g

h

kb -7.9

Ylp5

--5.7 --5.0 --4.5

--3.2 --2.8 --2.7

--2.5

2.0

1.6

Fig, 4. Restriction enzyme digests of pCFK8 and pCFKI6. Samples were digested with EcoRI and/or Hindlll and electrophoresed on 0.7% agarose gels as described in Materials and Methods. Lane (a) EcoRI-cleaved YIp5; Lane (b)EcoRl.-cleaved pCFK 16; Lane (c)EcoRI plus HindlII-cleaved pCFK 16; Lane (d) HindllI-cleaved pCFKS; Lane (e) EcoRI plus HindlII-cleaved pCFK8; Lane (f) EcoRI-cleaved pCFK8; Lane (g) Hindlll-cleaved Ad5 viral DNA and Lane (h)HaellI..cleaved M 13 RF (only fragments of 2.5 and 1.6 kb can be seen on this gel).

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luciliae maxicircle D N A p u b l i s h e d by H o e i j m a k e r s et al. [ 18]. F i n a l l y , the ars element has been localized to a 2 kb H i n d l I I - E c o R I f r a g m e n t derived from the 8 kb H i n d l I I fragment. This 2 kb f r a g m e n t c o r r e s p o n d s to the D3-R~ f r a g m e n t o n the published maxicircle map. This region of the maxicircle D N A has been f o u n d to hybridize very strongly to the 6 kb E c o R I f r a g m e n t RR2 o n the T. brucei maxicircle [18]. The recent f i n d i n g of ars activity associated with this 6 kb f r a g m e n t [19] suggests that C. fasciculata a n d T. brucei maxicircle ars elements m a y be related, A d d i t i o n a l insight m a y be provided by D N A sequence analysis of s u b - c l o n e d ars from each organism. ACKNOWLEDGEMENTS We are indebted to R a y m o n d K i m a n d Steve Bayne for frequent discussions a n d assistance. This work was s u p p o r t e d by a grant from the W o r l d Health O r g a n i z a t i o n a n d b y a p o s t d o c t o r a l t r a i n i n g g r a n t (to R.K.) in T u m o r Cell Biology from the N a t i o n a l C a n c e r Institute ( C A 09056). REFERENCES 1 2 3 4

5 6

7 8 9 l0 I1

12 13

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Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168. Kaguni, J. and Ray, D.S. (1979) Cloning of a functional replication origin of phage G4 into the genome of phage MI3. J. Mol. Biol. 135, 863-878. Southern, E.W. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick-translation with DNA polymerase I. J. Mol. Biol. 113,237-251. Hoeijmakers, J.H.J., Schoutsen, B. and Borst, P. (1982) Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidiafasciculata. I. Sequence evolution and transcription of the maxicircle. Plasmid 7, 199-209. Davison, J. and Thi, V.H. (1982) The Trypanosoma brucei maxicircle DNA contains ars elements active in Saccharomyces cerevisiae. Curr. Genetics 6, 19-20.